The lack of methods for efficient gene delivery to specific cell types remains a technical barrier to clinical use of gene therapy. In theory, it can be obtained by inserting ligands into retroviral Env to target infection to specific cell types that express cognate receptors for the ligands. In practice, hybrid Env directs specific binding but is unable or very poorly capable of gene transfer because of a post-binding defect. In addition, their subunit association was very unstable, leading to loss of hybrid binding subunit. Our goal is to develop improved hybrid Env for use in clinically relevant vectors that transduce specific cells efficiently. We will focus on overcoming their post-binding/fusion defect. We hypothesize that the fusion defects can be overcome by altering the Env sequences in two ways - adding compensatory mutations that increase infection by 1,500- fold or greater, or fusion-enhancing mutations that increase the overall fusogenicity of Env. Because the two compensatory mutations also stabilize hybrid subunit association, we will address this problem as well. Specific Aim 1 will test the hypothesis that the addition of one or more of the Env mutations can substantially increase infection via the cognate receptor. Infection and binding will be quantified using ligands that target receptors expressed on human tumor cells, but absent or expressed at very low levels on normal human tissues. The influence of ligand size, receptor density, or internalization of ligand-receptor complexes on hybrid Env-mediated infection will be determined. We will also determine the mechanism of action of mutations that increase infection, and the nature of the block remaining in hybrids that show little or no increased infection upon addition of mutations, by measuring the rate of lipid mixing and the ability to form fusion pores. In Specific Aim 2 a selective genetic method to isolate additional compensatory mutations will be developed and novel mutations isolated. New mutations might synergize the existing ones to increase infection further, and/or increase infection using hybrids that were not improved by the existing mutations. The mechanism of action of these mutations will also be determined.